CN107149588A

CN107149588A - Cosmetic composition

Info

Publication number: CN107149588A
Application number: CN201710316756.0A
Authority: CN
Inventors: 昂里克·阿拉巴塔; 帕特里夏·S·保
Original assignee: Restorsea LLC
Current assignee: Restorsea LLC
Priority date: 2012-01-23
Filing date: 2013-01-23
Publication date: 2017-09-12
Also published as: HUE030968T2; WO2013112569A1; EP2691069B1; EP2691069A1; US20140072547A1; AP2014007818A0; KR20130133285A; US8992996B2; EA201491325A1; AU2013212251A1; JP2015052002A; ECSP13013028A; CL2014001924A1; CA2829932A1; TN2013000473A1; CN103687586B; MX2014008804A; NI201400010A; KR101643556B1; BR112014017913A8

Abstract

The invention provides a kind of composition, it includes fish-egg Protein Separation thing.Wherein there is also the natural extracts containing unrighted acid and sterols material.A kind of emulsifying agent is additionally provided to form the mixture of the isolate and the extract.Present invention also offers a kind of composition, its antimicrobial comprising egg hatching Protein Separation thing and at least one protection isolate activity.Emulsifying agent has an aqueous phase being buffered between pH value 5.6 to 7.9 with isolate formation mixture, the mixture.Present invention also offers a kind of method for producing such cosmetics, this method includes forming an emulsion or an aqueous phase, and before isolate is added into emulsion, emulsion or aqueous phase are buffered to pH value between 5.5 to 7.9.Improve the method for skin appearance present invention also offers a kind of, this method include weekly at least three times by this it is used for cosmetic in skin to realize the improvement of skin appearance.

Description

Cosmetic composition

It is that on January 23rd, 2013, application number are that 201380001136.8, invention and created name is the applying date that the application, which is, The divisional application of the application for a patent for invention of " cosmetics ".

The cross reference of related application

The application is non-provisional application, it is desirable to the 61/589th, No. 592 U.S. Provisional Application that on January 23rd, 2012 submits Priority, the content of the provisional application is incorporated herein by reference.

Technical field

Present invention relates in general to cosmetics and its component, and more particularly to contain be conducive to improve skin quality The change of the egg hatching protein extract (egg hatching proteinaceous extracts) and its formula of (skin quality) Cosmetic.

Background technology

Traditional cosmetic formulations are contained to be formed protective coating to alleviate the influence that weather is caused on skin Oily substance and a variety of lipophilic substances.The suncream contained in cosmetics can also mitigate skin is caused with aging and freedom The relevant ultraviolet injury of generation of base.Other conventional ingredients of cosmetic composition are can to transport the water of considerable amount to skin The amount of moisture that the combined effect of the emollient (emollients) of skin, these water and oil discharges or retained from skin is to cover wrinkle Line.Regrettably, the formula of these Traditional makeups can not provide long-term for skin quality, the improvement in terms of chemistry.

The hand of the workman of fish hatchery has particularly preferred skin quality, even if contact cold water for a long time, this observation is separation The cause of a class novel protein related to fish-egg.For example in patent U.S.6,346,245, U.S.6,592,866, This proteinoid is described in detail in U.S.2011/0280882, WO2011/06434 and U.S.2009/0274770. The protein of this kind of separation includes the interior Pepsin (zonase) of fish-egg, leucoagglutinin (leukolectin), extremely acid It is at least some in treatment autoimmunity in acidic protein (VAPS) and certain cytase (choroelysins), these albumen Property disease and diseases associated with inflammation in terms of it is effective as treating damaging skin, have minimum side effect.Although this albumen The impaired skin function related with a variety of environmental stress factors to slacking is improved, it is such as dirty exposed to air-conditioning or air Thing is contaminated, these protein seem on the impaired very little or none influence of other properties affects related to the skin of aging.In addition, Although people once envisioned adds this fish-egg protein isolate (fish spawn isolate proteins) in cosmetics, But it is a technological challenge that protein active can be kept in cosmetic formulations and bin stability is given.

Accordingly, it is now desired to which a kind of cosmetics for the stable storing for solving the problems, such as a variety of skin agings, the cosmetics are whole Ovum and the particularly anti-inflammatory property of fish-egg derived protein are closed.Further, in addition it is also necessary to such cosmetics, its substantially free of There is unexpected toxic action to skin and have the synthesis cosmetic composition of biological cylinder accumulation problem.

The content of the invention

The invention provides a kind of composition, it comprises the fish-egg Protein Separation for having antiinflammatory action to mammal skin Thing (fish spawn protein isolate).Wherein there is also contain unrighted acid and sterols material (sterols) natural extracts, the natural extracts (natural product extract) have anti-oxidant Activity.The invention provides emulsifying agent or solubilizer, or combinations thereof to form the mixture of isolate and extract.This hair Bright to additionally provide a kind of composition, it comprises the egg hatching Protein Separation thing with the antiinflammatory action to mammal skin (egg hatching protein isolate).Wherein there is at least one antimicrobial (biocide), it protects ovum to incubate The activity for changing Protein Separation thing existsUp to 3 months shelf lifes, although applied when after egg hatching Protein Separation thing storage When subjects skin, the activity is maintained within the 80% of un-formulated and fresh protein isolate.Emulsifying agent is incubated with ovum Change Protein Separation thing formation mixture, the mixture has an aqueous phase being buffered between pH value 5.6 to 7.9.The present invention is carried The cosmetics box set for including above-mentioned composition is supplied, the box set uses the multi-purpose package of non-sterile, enclosed it applied to skin Skin is to promote the specification of skin renewal.

It is a kind of to produce the method containing the cosmetics to the active egg hatching Protein Separation thing of mammal skin, the party Method includes forming an at least aqueous phase and an at least hydrophobic phase.Emulsifying agent and at least one antimicrobial be present in this at least one In an at least phase in aqueous phase and an at least hydrophobic phase.An at least aqueous phase and this at least one it is hydrophobic it is combined with formed one breast Liquid.The emulsion or an at least aqueous phase are buffered to pH value between 5.5 to 7.9, are added by the egg hatching Protein Separation thing To before the emulsion.

Improve the method for skin appearance, including the skin by said composition for mammal the invention provides a kind of, Usage amount level is skin 1 × 10 every square centimeter^-6Mg to 0.1mg isolate and 2 × 10^-7Mg to 0.02mg unsaturation Aliphatic acid, weekly using at least three times, to realize the improvement of skin appearance.

Embodiment

The present invention has practicality as the cosmetics with skin repair characteristic.In one embodiment, an invention Cosmetics, which are formulated into, can provide the synergy between various ingredients.In another embodiment, the cosmetics group of an invention Compound, which is formulated into, does not contain synthesis component.The cosmetics of invention in all embodiments keep the activity of protein component, together When with bin stability at room temperature, and tackle because of non-sterile processing and by the microorganism pollution institute band caused by The Microbial Challenge come.The invention provides the production method of formula, this method produces a kind of use having in cosmetics With the activity and the cosmetic formulations of these characteristics of bin stability that protein is kept under condition of storage.

According to the present invention, egg hatching activated protein, the fish-egg point for particularly having antiinflammatory action to mammal skin living From albumen (fish spawn isolated protein), its with it is natural containing unrighted acid and at least one sterol Product extract is combined.The extract has antioxidation activity and collagen enzyme inhibition activity simultaneously, and it contains with that can make There is the emulsifying agent of the stabilized with mixture of Protein Separation thing and extract combined.It is to be appreciated that can be used in the present invention multiple Extract with reach composition and activity in terms of requirement.It has surprisingly been found that the activity of egg hatching albumen, and particularly fish-egg egg White and natural extracts act synergistically, to realize more preferable skin appearance and one in some embodiments of the invention The renewal of skin is realized in a little embodiments, compared with any composition is used alone in comparative examples.In other embodiments, originally The cosmetics of invention existUnder the conditions of there is the storage stability of at least three month, but when cosmetics are stored after for by During examination person's skin, the activity of the protein component is remained within the 80% of un-formulated and fresh protein isolate.

Fish-egg Protein Separation thing, it may for example comprise interior Pepsin (zonase), the leucoagglutinin of fish-egg (leukolectin) or combinations thereof, its property is in United States Patent (USP) U.S.6346245, the row-the of the 4th column the 15th 5 column eighth rows；[0321]-[0326] section and United States Patent (USP) U.S.2011/ in United States Patent (USP) U.S.2009/0274770 A1 [0157]-[0194] section is described in detail in 0280882；Crude extract containing this albumen；And combinations thereof.It is adapted to In Protein Separation fish-egg Typical sources include sturgeon (sturgeon), salmon (salmon), vendace (whitefish), The fish-egg of white trout (vendace), cod (cod), capelin (capelin) and burbot (burbot).It may be appreciated It is that other sources for the egg protein that can be used in the present invention include ootheca (the amphibian egg of amphibian animal Cases), such as ootheca of tadpole (tadpoles) and newt (salamanders), the ootheca of reptile (reptilian egg cases) and the ootheca of poultry (fowl egg cases).Under normal circumstances, an ovum or spy It is not the gross weight for the cosmetic formulations that the fish-egg Protein Separation thing containing at least some above-mentioned protein accounts for the present invention 0.0001% to 10%, if the 0.01%~10% of the crude extract is activated protein.It is understood that the present invention's The amount of protein present in one cosmetics depend on for example including the compatibility with other components of another cosmetics and Factor including the degree of skin injury.

It should be appreciated that in there is provided number range example, the number range should not only include the end of the scope Point value, and the intermediate value of scope, are specifically included in the range of this and finally having with the scope Imitate the intermediate value of numerical value (last significant figure) change.For example, the number range from 1 to 4 should When including 1-2,1-3,2-4,3-4 and 1-4.

The composition of the present invention also contains a kind of natural extracts.The natural extracts include unsaturated fat Acid and sterol, its amount, which reaches, to be enough to provide the antioxidant activity and collagen enzyme inhibition activity beyond control 10%, these activity Detection be respectively adopted oxygen radical absorbability (ORAC) test and in the culture medium containing 1% natural extracts Lipid peroxidation tracer and use ultraviolet-B radiation, amount of radiation be 150 millijoule every square centimeter.R.L.Prior,et Al. etc..Hydrophilic and lipophilic oxidation resistance (the oxygen radical absorbability of blood plasma and other biological and food samples (ORAC_FL)) determine (J.Agric.Food Chem.) (2003) 51:3273-3279.It is understood that natural extract is logical Often contain mixture, it is unrighted acid, one or more sterols and others have different skin disease to learn activity The mixture of (dermatological activity) composition.The product extract used in a composition of the invention Also collagenase activity can be suppressed, suppressed in certain embodiments up to more than 5%, the numerical value is detected by the comparison with control group Obtain, the extract containing 0.1% percentage by weight in control group, the detection uses the range estimation operational procedure of anticollagenase, right The biopsy of cold skin section with having cultivated 3 hours at 37 DEG C detected, cold skin section and and Do not cultivated together with extract, then the culture not with (negative control) or with every milliliter of 15 microlitres of clostridiopetidase As together at 37 DEG C Three hours, are the rinsing of biopsy sample afterwards, afterwards to trichome stain (trichome of Masson) mesh Survey is dyed and microexamination.In certain embodiments, natural extracts bag used in composition of the invention Include at least one of ω -3, ω -6 and ω -9 aliphatic acid.In other embodiments, unrighted acid is in natural product extraction Amount present in thing is at least 20 milligrams in every 100 grams of extracts.The natural extracts of the present invention also include at least one The sterol of type, such as sterol include cholesterol, campesterol, stigmasterol, sitosterol, and its hydroxylated form and they Combination.In certain embodiments, the amount that at least one sterol is present in the extract of natural products is every 100 grams of extracts In be more than 50 milligrams.Generally, in composition of the invention natural extracts weight for composition total weight 0.01~ 10%, 0.1~3% for gross weight in certain embodiments.Determine the factor in terms of the amount of selection natural extracts, example Such as stability of egg hatching albumen and glucosides including cosmetics in the presence of egg hatching albumen, the degree of skin lesion and cosmetic The shelf-life of product.

It is easy to prepare by routine techniques according to the natural extracts of the present invention, described routine techniques is for example Pass through one of hydrophily or the hydrophobic first solvent extraction such as plant of leaf, stem, bud, root, fruit or combinations thereof Divide and the mixture is filtered after contacting 1-48 hours with the solvent, the amount of the solvent is usual relative to plant material 1-20's Volume ratio.Extracted in hydrophilic organic solvent is reduced and eluted to obtain the day of the present invention by routine techniques Right product extract.The extractive technique of representational aliphatic acid/sterol refers to J.S.O ' Brien and A.A.Benson, J.Lipid Res.,5, 1964,pp.432-436..The organic solvent, such as including ethers ethers, furans furans, Petroleum distillate petroleum distillates, alkane (alkanes), halogenated alkane (halogenated alkanes), alcohol Class (alcohols), cellulose acetate (acetates), and combinations thereof.Fully interact usual 20 between each phase Minute to after 48 hours, by organic phase separation to obtain the extract according to the present invention.The suitable source of extract is included in this The red algae of " marine alga ", green alga, brown alga and tufted cyanobacteria (tuft-forming are all referred to as in text ), and plant or theirs have purple, red, the composition of the coloring of black or orange fruit or leaf cyanobacteria Part, leaf colour chlorophyll it is present or absent under the conditions of measure.Seaweeds represents containing with height for the present invention The common denominator of the unrighted acid of amount and the natural extracts of sterols material.It is to be understood that unsaturated lipid Fat acid and the synthesis of sterols material and/or animal origin are easily used in the composition of the present invention.

Except egg hatching Protein Separation thing and natural extracts, the present composition in some embodiments is contained It is usually used in one or more of cosmetics other compositions, precondition is that these compositions maintain egg hatching Protein Separation thing and day The property of right product extract.The representational Traditional makeup composition in the cosmetics of the present invention is easily formulated into, For example including the deionized water for the most of weight that may be constructed cosmetics of the present invention；Such as oil (petroleum), lanolin (lanolin), ceresine (ceresin), microwax (microcrystalline wax), Brazil wax (carnuba wax), C₁₀-C₃₀Aliphatic acid, C₁₀-C₃₀The solid and Wax (solids and waxes) of alcohols material；Such as olive oil (olive oil), jojoba oil (jojoba oil), castor oil (castor oil), sunflower oil (sunflower oil), hazel Seed oil hazelnut oil, shea butter (shea butter) and safflower oil (safflower oil) oily substance；Such as Guar gum (guar gum), xanthans (xanthan gum), carrageenan (carrageenan), hydroxyethyl cellulose (hydroxyethyl cellulose), fungi plant glue (sclerotium gum), carbomer glue (carbomers), poly- third Olefin(e) acid ester (polyacrylates) and acrylate-C₁₀-C₃₀Alkyl acrylate copolymer (acrylate-C₁₀-C₃₀alkyl Acrylate copolymers) viscosity modifier/stabilizer；Such as glycerine (glycerin), propane diols (propanediol), sodium lactate (sodium lactate), the hydrophily NMF of Anjidew NL50 (sodium PCA)；Such as two is pungent Base ether (dicaprylyl ether), myristyl myristate (myristyl myristate), C₁₀-C₃₀Caprate (C₁₀-C₃₀Caprates), the C containing octanoic acid and/or capric acid₁₀-C₃₀Triglycerides and C₁₀-C₃₀Glyceride emollient； Such as hexadecanol (cetyl alcohol), octadecanol (stearyl alcohol), tristerin glyceryl Sterate, stearic acid (stearic acid), glycol glycerin ester (glycol sterate), bi-tristearin The assistant for emulsifying agent of (glyceryl distearate)；The breast of such as nonionic surface active agent and anion surfactant Agent, salt, the Caproyllactic acid of described anion surfactant for example containing 2-Lauroyloxypropionic acid (lauroyl lactylate) Salt, salt, the stearoyl lactylates (stearoyl of cocounut oil acyl lactic acid (cocoyl lactylate) of (caproyl lactylate) Lactylate salt, the salt of olive oil spermaceti alcohol ester (cetearyl olivate), olive oil D-sorbite (sorbitan) Olivate salt or the salt of glyceryl oleate citrate (glyceryl oleate citrate)), these salt it is typical Cation includes sodium ion, potassium ion, ammonium ion, calcium ion and magnesium ion；Silicone oil material；It is such as inorganic auxiliary with thing (inorganic accompaniments) and inorganic dyestuff colouring agent；It is inorganic or organic that metal saponification process or silicon are handled Pigment and organic dyestuff；Nonionic surface active agent；Polysiloxanes (polysiloxanes)；Water preservative (water preservatives)；PH adjusting agent；The vitamin and vitamin of such as vitamin E, vitamin K or B family vitamin compound Metabolin；Essential oil (essential oils)；Absorb compound (the ultraviolet light absorbing of ultraviolet light compounds)；Aromatic (fragrances)；Wetting agent (humectants)；Blood flow reinforcing agent (blood flow enhancing agents)；Induce the material (agents inducing a cold sensation) of creeping chill；Antiperspirant (antiperspirants)；Active material (actives) and antimicrobial.

Active material used herein be defined as being applied to application on human skin to clean, beauty, improve attraction or improve outer The organic compound of sight, the organic compound have improve skin properties effect, no matter the property of the compound be polarity or It is nonpolar.The term used herein, especially excludes egg hatching albumen, the mixture of egg hatching albumen and natural products and carries Take thing.The acitve organic matter matter that may be used herein, such as comprising extra extract, such as the extract of following material：Mulberry Scape mushroom (mushroom mpo Songyi), Acacia (Acacia farnesiana), the Brazilian certain kind of berries (Euterpe Oleracea), bright leaf Malpighia coccigera (Malpighia glabra) , Rouge fat trees (Bixa orellana), dessert almond (Prunus Dulcis), aloe (Aloe vera), high mountain rose azalea (Rhododendron ferrugineum), amaranthus caudatus (Amaranthus caudatus), root of Dahurain angelica root (Angelica archangelica), anise (Pimpinella anisum), Apple (Malus domestica), apple peppermint (Mentha suaveolens), oat (Oat avenanthramide), Almond -apricot (Prunus armeniaca), Montana Arnica (Arnika montana), cynara scolymus (Cynara scolymus), Asparagus (Asparagus officinalis), avocado (Persea americana), cardiospermum halicacabum (Cardiospermum Halicacabum), lemon balm (Cardiospermum halicacabum), yushania (Bambusa vulgaris), powder fragrant plant Any of several broadleaf plants (Musa paradisiaca), monkey-bread tree (Adansonia digitata), thorn bark of a cork tree (Berberis vulgaris), sieve Strangle (Ocimum basilicum), bay (Laurus nobilis), willow herb (Epilobium angustifolium), ramps (Allium ursinum), Eurasian yellow avens (Geum urbanum), European birch (Betula pubescens), Su Linnan is bitter Wooden (Quassia amara), fennelflower (Nigella sativa), blackcurrant (ribes nigrum), black mulberry (Morus Nigra), radish (Raphanus sativus), wild tea tree (Camellia sinensis), European black berry (Rubus Fruticosus), Iris versicolor (Iris versicolor), whortle (Vaccinium myrtillus), Common Borage (Borago officinalis), broad bean (Vicia faba), buckbean (Menyanthes trifoliata), buckwheat (Fagopyrum esculentum), burdock (Arctium lappa), butcher's broom (Ruscus aculeatus), cocoa (Theobroma cacao), calamus (Acorus calamus), Caraway (Carum carvi), cardamom (Elattaria Cardamomum), carrot (Daucus carota sativus), Ka Tuba coca (Erythroxylum catuaba), hundred Golden flower (Centaurium erythraea), chamomile (Matricaria chamomilla), cherry (Prunus Cerasus), chick-pea (Cicer arietinum), chlorella (Chlorella vulgaris), Black Box Tracing (Aronia melanocarpa), Peruvian bark tree (Cinchona pubescens), Ceylon cinnamon (Cinnamomum Verum), cloves (Syzygium aromaticum), coconut (Cocos nucifera), Coffea arabica (Coffea Arabica), Coleus forskohlii (Plectranthus barbatus), coltsfoot (Tussilago farfara), comfrey (Symphytum officinale), greater plantain grass (Plantago major), coriander (Coriandrum sativum), corn (Zea mays), corn poppy (Papaver rhoeas), corn flower (Centaurea cyanus), cotton (Gossypium Herbaceum), blueberry (Vaccinium vitis-idaea), cowslip (Primula veris), Cranberry (Vaccinium macrocarpon), cucumber (Cucumis sativus), turmeric (Curcuma longa), daisy (Bellis perennis), Damask Rose (Rosa damascena), damiana (Turnera diffusa), the West Dandelion (Taraxacum officinale), nipa palm (Phoenix dactylifera), white dead nettle (Lamium album), Dog rose (Rosa canina), dragon fruit (Hylocereus undatus), Sanguis Draxonis (Daemonorops draco), narrow leaf Echinacea purpurea (echinacea angustifolia), edelweiss (Leontopodium alpinum), golden elder (Sambucus Nigra), wilsonii (Acanthopanax senticosus), emblic (Phyllanthus emblica), Blue gum (Eucalyptus globulus), oenothera biennis (Oenthera biennis), psammophytes strawflower (Helichrysum Arenarium), eyebright (Euphrasia rostkoviana), fenugreek (Trigonella foenum-graecum), nothing Flowers and fruits (Ficus carica), flax (Linum usitatissimum), frangipanis (Plumeria alba), cone purple Viola (Fumaria officinalis), garden sorrel (Rumex acetosa), gardenia (Gardenia Yessminoides), Huanglong Courage (Gentiana lutea), ginger (Zingiber officinale), ginkgo (Ginkgo biloba), ginseng (Panax Ginseng), activities of Solidago virgaurea L (Solidago virgaurea), grape (Vitis vinifera), grape fruit (Citrus Paradisi), wild tea tree (Camellia sinensis), Brazil's perfume cocoa (Paullinia cupana), hay flower (Graminis flos), european railways (Corylus avellana), garden balsam (Lawsonia inermis), roselle (Hibiscus sabdariffa), honeysuckle (Lonicera japonica), hops (Humulus lupulus), Europe Horse chestnut (Aesculus hippocastanum), meadow pine (Equisetum arvense), long-living flower (Sempervivum Tectorum), Hyssopus officinalis (Hyssopus officinalis), Iceland's lichens (Cetraria islandica), counterfeit indigo (Baptisia tinctoria), Irish moss (Chondrus crispus), ivy (Hedera helix), element virtue flower (Jasminum officinale), Jojoba (Simmondsia chinensis), Juniperus communis (Juniperus Communis), lady's-finger (Anthyllis vulneraria), African pendent lamp tree (Kigelia africana), macaque Peach (Actinidia chinensis), the root of kudzu vine (Pueraria lobata), garment or robe made of feathers grass (Alchemilla vulgaris) is smoked Clothing grass (Lavandula angustifolia), lemon (Citrus limon), lemongrass (Cymbopogon cytratus), light Fruit radix glycyrrhizae (Glycyrrhiza glabra), madonna lily (Lilium candidum), carrys out lemon (Citrus Aurantifolia), leaflet linden (Tilia cordata), lotus (Nelumbo nucifera), Queensland nut (Macadamia Ternifolia), Y biondii(Pamp)D.L.Fu (Magnolia biondii), leaf of Chinese ilex barberry (Berberis aquifolium), high mallow (Malva sylvestris), mango (Mangifera indica), pot marigold (Calendula officinalis), Europe Chestnut (Castanea sativa), medicine hollyhock (Althaea officinalis), rotation fruit palmate meadowsweet (Filipendula Ulmaria), muskmelon (Cucumis melo), milk thistle (Silybum marianum), broomcorn millet (Panicum miliaceum), Laughable beans (Colophospermum mopane), Sorbus alnifloria tree (Sorbus aucuparia), feltwort (Verbascum Thapsus), Siberian elm leaf Chinese parasol tree (Guazuma ulmifolia), myrtle (Myrtus communis), nasturtium (Tropaeolum majus), margosa tree tree (Azadirachta indica), stinging nettle (Urtica dioica) steeps leaf Algae (Ascophyllum nodosum), nutmeg (Myristica fragrans), summer oak (Quercus robur), palmate sea Band (Laminaria digitata), oat (Avena sativa) must sunglo (Usnea barbata), olive (Olea Europaea), onion (Allium cepa), sweet orange (Citrus sinensis), lemon-mint (Mentha citrata), ox To (Origanum vulgare), heartsease (Viola tricolor), papaya (Carica papaya), capsicum (Carica papaya), Sheep's-parsley (Petroselinum crispum), passionflower (Passiflora incarnata), flowering peach (Prunus persica), European pear (Pyrus communis), centella (Centella asiatica), Chinese herbaceous peony (Paeonia officinalis), pepper (Piper nigrum), lavender (Mentha piperita), pineapple (Ananas Comosus), cherry, European Lee (Prunus domestica), pomegranate (Punica granatum), jerusalemsage (Pulmonaria officinalis), cucurbita pepo (Cucurbita pepo) , Wen Quinces (Cydonia oblonga), raspberry (Rubus idaeus), red clover (Trifolium pratense), paddy rice (Oryza sativa), wire A Sibala is appropriate (Aspalathus linearis), Rosa centifolia (Rosa centifolia), rosemary (Rosmarinus officinalis), Rye (Secale cereale), Salvia japonica (Salvia officinalis), the fruit of Chinese magnoliavine (Schisandra chinensis), Sea-buckthorn (Hippophae rhamnoides), sesame (Sesamum indicum), shepherd's purse (Capsella bursa Pastoris), Flos Albiziae (Albizia julibrissin), alkali wormwood artemisia (Artemisia abrotanum), spearmint (Mentha spicata), wheat (Triticum aestivum), blunt top spirulina (Spirulina platensis) is moved Prestige dragon spruce (Picea abies), hypericum perforatum (Hypericum perforatum), carambola (Averrhoa carambola), STEVIA REBAUDIANA (Stevia rebaudiana), strawberry (Fragaria ananassa), sunflower (Helianthus annuus), Daghestan sweet clover (Melilotus officinalis), Dalmatian chrysanthemum (Tanacetum cinerariifolium), citrus (Citrus Reticulata), thyme (Thymus vulgaris), Chinese lime rattan (Salvadora persica), valerian (Valeriana Officinalis), vanilla (Vanilla planifolia), Verbena officinalis (Verbena officinalis), sweet violet (Viola odorata), walnut (Juglans regia), Nasturtium officinale (Nasturtium officinale), watermelon (Citrullus lanatus), wheat (Triticum aestivum), wild tea tree (Camellia sinensis), hawthorn (Crataegus rhipidophylla), safflower thyme (Thymus serpyllum), white willow (Salix alba), Fu Ji Buddhist nun Asia state witch hazel (Hamamelis virginia), Radix Isatidis (Isatis tinctoria), lycium barbarum (Lycium Barbarum), absinth (Artemisia absinthium), Europe kind grass (Achillea millefolium), the red wingceltis in South America (Hymenaea courbaril), Cananga odorata (Cananga odorata), Trichotoma matsutake (Tricholoma matsutake), or Combinations thereof.It is appreciated that these extracts also have a certain degree of antibacterial activity, strengthen anti-micro- in certain embodiments Effect of biological agent.The organic compound of activity is normally used in the formula of the present invention, accounts for the total formula weight of the present invention 0 to 1%, up to the 5% of gross weight in certain embodiments.Although these active materials are often to be more tolerant of traditional cosmetic Product processing conditions, and in many cases, different from egg hatching albumen and natural extracts, it is adapted to be contained in aqueous phase Or in lipophilic phase, it is to be understood that these active organic compounds in certain embodiments, by a C phase or other, After emulsion formation, it is added separately in emulsion.

The cosmetics of the present invention are easy to be configured to aqueous cosmetic (water-based cosmetic), such as including skin toner Water (toner), sprays (spray mist), Essence (serum), face cream (cream), facial mask (mask), suncream (sunscreen), clear face is newborn (cleanser), moisturiser (moisturizer), bath configuration product (bath Preparation), foundation emulsion (liquid foundation), astringent (astringent), shaving product (shaving Preparation), hair products (hair care product), and other skin care item, or topical agent, for example including Antiphlogistic.There is provided a kind of cosmetics in only certain exemplary embodiments of this invention, its vegetalitas comprising such as lactate is emulsified Agent (plant based emulsifier), the vegetalitas emulsifying agent, substantially free of silicones (silicone-free), bath Mineral oil is free of with configuration product, without paraffin oil (petrolatum), and without the paraben preservative of synthesis (parabens).These compounds of emulsifying agent substantially free are defined here as these compounds and accounted for altogether less than 10% The percentage by weight of emulsifying agent, formula of the invention is easily applied to the skin of people, particularly and for example including whole face Region, mouth, circumocular region, forehead, temples, cheek or chin；Hand；Pin；Neck；Scalp；Ancon；Chest；Forearm；Shoulder Wing；Leg；And combinations thereof.

The cosmetics of the present invention presently, there are the active difficulty for being difficult to that protein is kept in process for preparation.It is general next Say, it can be seen that heating and high shear mixing, the routine techniques of these cosmetic formulations are often denatured activated protein.Furthermore, Easily there is microbial degradation (microbial degradation) in activated protein, particularly when the cosmetics of the present invention be Processed under conditions of non-sterile, andStorage at room temperature when.The formula of the cosmetics of the present invention needs preparing PH value, shear-mixed and the environment of heating in journey by egg hatching albumen in limited scope, to keep wherein existing Egg hatching albumen activity.In order to keep egg hatching protein active, cosmetic formulations process includes constituting in all cosmetics PH value is mutually buffered between 5.6 and 8, before egg hatching albumen is added into that phase.Being buffered in this area often makes With, and including addition acid, alkali, sour salt, or combinations thereof to balance the aqueous solution or aqueous phase of emulsion to desired pH value. It is understood that it is routine techniques that pH, which is determined, quantitative determination can pass through volumetric precipitation method (volumetric ) or such as can be from Thermo Fischer Scient Inc. (Thermo Fisher Scientific) (Waltham titration MA, USA) purchase potential electrode (potentiometric electrodes)；Observational measurement uses litmus paper kit.

Although in order to keep the activity of albumen, the pH value of the cosmetics of the present invention is adjusted between 5.6 and 7.9, part Suppress the microbial degradation of egg hatching albumen, but antimicrobial is present in cosmetic formulations of the present invention to keep protein Activity, for the microorganism brought in process for preparation and for during using multi-dose container, through subject's part The cosmetics of the contact present invention with breathing, the situation that various microorganism continuous are constantly inoculated with cosmetics.Therefore, this hair Bright multipurpose cosmetic formulations and the formula of stable storing, the combination comprising broad-spectrum antimicrobial agent or antimicrobial.This The antimicrobial of the wide spectrum at place is defined as, and it has resisting gram-positive bacteria, Gram-negative bacteria, and and healthy human body The activity of related fungi.Effective antimicrobial herein, such as including benzoic acid (benzoic acid), para hydroxybenzene first Esters of gallic acid (parabens), salicylic acid (salicylic acid), carbolic acid (carbolic acid), sorbic acid (sorbic Acid), alkyl p- p-hydroxybenzoates (alkyl p-hydroxybenzoates), to chloro metacresol (p- Chlorometacresol), hexachlorophene (hexachlorophene), benzalkonium chloride (benzalkonium Chloride), chlorination chlorhexidine (chlorohexidine chloride), trichloro-symmetrical diphenyl urea (trichlorocarbanilide), phenoxetol (phenoxyethanol), acyl group methyl amimoacetic acid (acylsarcosines), glutathione (glutathione), malic acid (malic acid), tartaric acid (tartaric Acid), ascorbic acid (ascorbic acid), ascorbate (ascorbates), it is necessary to vegetable oil, become chain fibroin (mutacin proteins), and combinations thereof.In some embodiments of the invention, the anti-micro- of natural origin is only existed Biological agent.The antimicrobial of natural origin, such as including such as lactobacillus (Lactobacillus), streptococcus mutans (Streptococcus mutans) and the ferment filtrate of leukonid (Leuconostoc)；Bisabol (bisabolol)；Eucalyptus Olein (eucalyptol)；Thymol (thymol)；Inositol (inositol)；Saponin；Such as Japanese honeysuckle (Japanese honeysuckle, Lonicera japonica), sheep hoof (yangti (Rumex japonicus)), kuh-seng (kushen (Sophora flavescens)), water lily (candock), wild wild marjoram (wild oregano), orange (orange ), Salvia japonica (sage), Manny Buddhist (manifoil), high mallow (common mallow), Ligusticum wallichii (chuanxiong (Cnidii Officinale Makino)), Japanese green gentian (Japanese green gentian (Swertia japonica Makino)), bisabol (bisabolol), thyme (thyme), Radix Angelicae Sinensis (danguii (Angelica sinensis)), orange Skin (orange peel), birch (birch), meadow pine (field horsetail), sponge gourd (dishcloth gourd), seven leaves Set (horse chestnut tree), kiss-me (creeping saxifrage (Saxifrage stolonifera)), mountain Golden car (arnica), lily (lily), argy wormwood (mugwort), tree peony (peony), aloe (aloe), gardenia And those are in document M.M.Cowan, Clinical Microbiology Reviews, 12 (4) Oct.1999, (gardenia) P.564 the natural extract for the plant being described in detail in -582, or combinations thereof.These extracts are commented by clinical microbiology Come by the step of detailed description in (Clinical Microbiology Reviews, 12 (4) Oct.1999, pages.573-574) It is made and generally uses a kind of such as C 1-C 8 alcohol (C1-C8alcohol), polyhydroxy-alcohol (polyhydric Alcohols), the hydrophilic organic solvent of water and aqueous alcohol (aqueous alcohols).In a specific present invention In embodiment, using Japanese honeysuckle (Japanese honeysuckle), phloretin (phloretin) (apple), galangal Plain (strawflower (Helichrysum aureonitens)) is each used alone or in combination as the antimicrobial of natural origin. It is to be understood that except bactericidal property, natural extract usually assigns a kind of fragrance of cosmetics of the present invention.Antimicrobial It is 0.1%~5% (weight) of gross weight in certain embodiments in the multifunctional packaging of the cosmetics of the present invention, can To be appreciated that, the amount more than 5% antimicrobial of gross weight easily can be put into as requested.

In certain embodiments, pH delays any value between 5.6 to 7.9 to the cosmetics of the present invention, and at other In embodiment, it is buffered between 6.5 to 6.8, but in other embodiments, is buffered between 6.8 to 7.3.It is not intended to Be limited to it is one specific theoretical, such as pH value promote such as Egg extracts of fish-egg isolate and the extract of natural products into The stabilisation and bin stability divided.By preparing at least two components in advance, the present invention is together promoted with optional additional phase The formation of the formula of cosmetics, described at least two components include emulsion oil-in-water and water-in-oil type or organic silicon emulsion Second phase, described additional phase, the active Egg extracts and/or natural extracts of isolate of for example being laid eggs containing such as fish Third phase.Or, it will be understood that egg hatching isolate or natural extracts one of which or both easily disperse In emulsion oil-in-water, water-in-oil emulsion, water-in-silicone emulsion or serum preparation.It was surprisingly found that adding Plus before active egg protein extract, various phase pH buffering is for ensureing the bin stabilities of final cosmetics of the invention It is vital with activity of the albumen on subjects skin.

The phase component of the fraction of emulsion oil-in-water, such as including oil, wax, ester, ether, butter, glyceride, or they Composition, as detailed above.The most phase component of emulsion oil-in-water, such as including water, water soluble salt (aqueous Salts), wetting agent and various water-soluble substanceses, as detailed above.Emulsifying agent for the system includes ionic surfactant Agent, nonionic surfactant as detailed above, and also includes lecithin, liposome and elaiosome (oleosomes).Its He can easily provide non-essential additive according to as detailed above, and it includes viscosity modifier, and emulsion stabilizer is fragrant Material, it is such.In the particular embodiment, the pH value of oil-in-water emulsion be 5.6 to 7.9 between.In some embodiments In, the pH value of emulsion oil-in-water is 6.5 and 6.8.

The relative quantity of the fraction and most phase component of water-in-oil emulsion or organosilicon water-in type emulsion can become Change, and preferred, it is used as emulsifying agent dependent on nonionic surface active agent and organosilicon derivates.Viscosity modifier and/ Or emulsion stabilizer, such as including water soluble salt, natural gum, wax, and their compositions, as detailed above.Specific real Apply in example, the pH of interior phase is adjusted between 5.6 to 7.9.In certain embodiments, interior phase pH value regulation 6.5 to 6.8 it Between.It is appreciated that various vitamins, such as vitamin E, the ascorbic acid derivates of vitamin K, such as ascorbic acid glucoside, One or two in above-mentioned Emulsion Phase can be easily added to, or is used as an independent third phase.

The cosmetics of the present invention can be formed easily by the combination of pre- mixed phase as detailed above, or optional , all these compositions are mixed to form a homogeneous mixture successively according to the conventional successive of this area.The invention provides It is a kind of to improve the method for skin appearance, including the composition of the present invention is used for the skin of mammal, consumption is every square li Rice skin 1x10^-6Mg to the 0.1mg isolate and 2x10^-7Mg to the 0.02mg unrighted acid, weekly at least Three times, to reach the improvement of skin appearance.

The typical range of cosmetics of the present invention is summarized as shown in table 1-3.It should be understood that as being described in detail herein, it is other Conventional basic components, are combined individually or with the natural extracts containing unrighted acid and sterol, can be very Easily use method detailed in this article to be adjusted to stablize egg hatching albumen.It is workable for the various of cosmetics herein Conventional basic components referring to：" cosmetic formulations ", the second edition, 2001 volume 8, Noyes publishing house (ISBN0-1855- 1454-9 )(E.W.Flick,“Cosmetic and Toiletry Formulations”,2^ndEd.,Vol.8；2001, Noyes Publications, Norwich,New York,USA(ISBN 0-1855-1454-9).)。

The typical range of the emulsion oil-in-water form of the cosmetics of the present invention of table 1., wherein percentage is the hundred of gross weight Divide ratio.The form of three-phase or four phases is provided herein.

Three-phase is formulated

A-1 phases：

B-1 phases

C-1 phases

Active (0.034% active egg hatching albumen) 0.001~10

Active (natural extracts) 0.001~10

Four are mutually formulated

A-2a phases

A-2b phases

B-1 phases

C-1 phases

Active (0.034% reactive protein) 0.001~10%

Active (natural extracts) 0.001~10%

*-pH regulations are carried out after can also being mixed after A phases and B, but will be before C-1 phases are added.

*-need the antimicrobial of some quantity, the multiduty non-sterile for generally accounting for gross weight 0.1-5% is matched somebody with somebody Side.

The typical range of the organosilicon water-in type emulsion form of the cosmetics of the present invention of table 2, percentage therein is gross weight The percentage of amount.

A phases

B phases

C phases

Active (0.034% reactive protein) 0.001~10%

Active (natural extracts) 0.001~10%

The typical range of the serum formula of the cosmetics of the present invention of table 3, percentage therein is the percentage of gross weight.

A phases

B phases

Solubilizer 0.1~10%

Nonpolar active organic compounds 0~1%

C phases

Active (0.034% reactive protein) 0.001~10%

Active (natural extracts) 0.001~10%.

Embodiment 1

The later of the formulated three-phase prepares a cosmetic cream (cosmetic cream).A phases are heated to 75-80 DEG C, and It is sufficiently stirred for forming uniform mixture.B phases are placed in an independent container at room temperature and are pre-mixed, and 40°

C or following temperature are added to A phases.The mixture of A phases and B phases obtained by phase C is added to successively, and fully Stirring is until forming uniform frost.Obtain uniform and stable frost.

Embodiment 2

There is provided a kind of cosmetic cream, wherein fish-egg Protein Separation thing and natural prodcuts extract is all the component of A phases, without It is as fish-egg Protein Separation thing and natural prodcuts extract are present in an independent C phase in each embodiment 1.By in room A phases are formed under temperature to avoid albuminous degeneration, have obtained a kind of similar frost.

Comparative example

The composition (Comparative examples A) being compared with embodiment 1 is prepared, it is free of fish-egg Protein Separation thing.Another group Compound is free of natural extracts (comparative example B).Also it is prepared for being not only free of fish-egg Protein Separation thing but also is carried without natural prodcuts Take the control formula (comparative example C) of thing.The seminar of 40 people is divided into 4 groups, and each group of people is daily using the frost or ratio of embodiment 1 Any one compared with example A-C is up to 12 week.The foundation elasticity of the skin of each subject is measured, and the research measured Conclusion be based on using comparative example C as reference standard, the composition of embodiment 1 shown with Comparative examples A-C compared with the superior degree of packing with Smoothness.All compositions are standardized to reach such level：1x10 is used on skin every square centimeter every time^-6In the least Gram isolate (if any) and 2x10^-7The unrighted acid (if any) of milligram.

Embodiment 3

Fresh, unfertilized salmon (Ontario salmon (Salmo salar)) ovum, it was obtained from (evening in reproduction period Autumn) female, be placed on ice, and preferably prepare extract immediately., can be with the case where not destroying egg membrane The ovum of freeze-drying in cryoprotector (such as 1.5M 1,2- propane diols and 0.2M sucrose).Freezing mode can be gradually Ground (- 1-C./min) is to-80 DEG C.During prepared by whole extract, ovum should be defrosted being placed on ice.

Ovum is placed on containing being washed twice in protease inhibitors (10 μ g/ml) HBSS or seawater.Cleaning solution is removed, by ovum Dissolve and carried out in the Dounce glass-glass homogenizer (Dounce glass-glass homogenator) of a precooling Homogenate.Lysate is transferred to the ultrapure polymer centrifuge tube of Beckman (Beckman Ultra Clear polymer Centrifuge tubes) (5ml), while avoiding being transferred to chorion, then in the Beckman centrifuge using SW55T1 rotors In, 4 DEG C of rotating speeds with 15,000g centrifuge 15min.Thus obtain 3 parts, top section lipid, center section cytoplasm and Contain the floor portions of chorion and fragment.Center section cytoplasm is extract to be collected.The extract will be estimated to be contained There are the organelle of most intracellulars, including mitochondria, lysosome and peroxisome, the extract should be limpid and viscous Thick, and have orange color.Protease inhibitors (10 μ g/ml stocks) is added, and extract is maintained at-80-C. The middle blastaea of the embryo of zebra fish is collected, liquid is removed, and be refrigerated to -20 DEG C.To prepare the extract, embryo is positioned over Thaw, dissolved on ice, and using Dounce glass-glass homogenizer (Dounce glass-glass homogenator) It is homogenized in a small amount of HBSS or seawater (more preferably less than 50% liquid v/v).The lysate is through a sterile flax Cloth is filtered, and in the Beckman X-22R centrifuges of SX4250 rotors are employed, 4 DEG C, is centrifuged with 5,000 rotating speed 20min.Collect the Cytoplasmic extract (supernatant), and addition protease inhibitors (10 μ g/ml).The extract can be micro- Filter (0.22 micron of MilliQ sterilizing filters) in hole.The extract is placed in -80 DEG C.The pH of the extract uses reindeer moss Test paper detects that its protein concentration is determined by Bradford detection methods, permeability is determined by osmometer.

The general program is applied to prepare the extract from sea urchin, shrimp, fish egg/ovum or frog ovum.Briefly say, it is pregnant The raun being pregnent just has given birth to ovum soon, generally at dawn (2-4AM), collects their ovum, and the pregnant raun is in spawning program In, the 6-8 hours before spawning inject hCG hormones (1ml/kg), or the frog from gestation.By ovum/egg freeze-drying Or be refrigerated to -20 DEG C or fresh use.Ovum is collected from different types of fish.For sea urchin, 0.5M KCl are expelled to face periphery To trigger coming off for ovum.The extract is prepared by the way that egg/ovum is crushed into (cell crushing instrument or dounce- homogenizers) or not Same centrifugation is to separate the cytoplasm of whole contents, with/without chorion (oolemma), with/without core/born of the same parents Liquid, with/without organelle, with/without lipid.Further fractionation can be carried out one or more to separate MRNA, protein, small peptide, carbohydrate and lipid.The main component of aliphatic acid in ovum is oleic acid, linoleic acid and ω -3 fat Fat acid.

Salmon Egg extracts are obtained using above-mentioned operation sequence, the salmon Egg extracts have very high protein Content, 100-380mg/ml, pH are between 6.4-6.8 for the protein content scope, and permeability is about 350mOsm.Should Extract should be limpid and sticky and non-filterable (the MilliQ filter by 0.45 micron).The extraction Protein in thing, because of its high protein content and low pH, it can be by adding water or aqueous molten with low-buffer capacity Liquid is deposited.Extract can adjust pH by adding basic species (species are extracted per ml and add 1-3 μ l 1M NaOH) Value is to 7.0, and therefore, it dilutes in water or aqueous solution is possible.The protein concentration scope 23- of zebra fish extracts 26mg/ml, pH are between 6.4-6.8, and permeability is in 80-150mOsm.The extract is limpid and not sticky, can Filtering and easily can be diluted in water with various dilution ratios.

Embodiment 4

The interior Pepsin (zonase) of the fish-egg for the salmon that gel filtration-purifying is obtained plus compatibility-purifying passes through One final chromatographic procedure can be further purified the purity for reaching sequence-level.The program is PBE94 pillars, Tris- acetate buffer solutions (10mM, pH 9.0), subsequent eluent is the buffering with salt gradient (up to 1M NaCl salt) Liquid.This step increases the catalytic activity of the interior Pepsin (zonase) of fish-egg up to the overall purifying for being higher by 7.6 times, 714 times, With the 28% of parent material yield.The purification step causes the protein characteristic of the interior Pepsin (zonase) of fish-egg to keep It is whole, it is used as a 28kDa half race (moiety).Therefore, the step is not prepared from the interior Pepsin (zonase) of the fish-egg Unrelated, the major protein pollutant of middle removal.For being present in artemia hatching solution and " the interior Pepsin (zonase) of fish-egg slightly production The race of interior Pepsin (zonase) half of fish-egg in product ", the molecular weight of the interior Pepsin (zonase) of the fish-egg of purifying with By Western blotting technology for detection obtain it is identical.

Obviously, it happens is that small, pollution polypeptide is removed in third time and last chromatographic procedure.This A little polypeptides carry the oligopeptides of many residues around seemingly, most probable is from chorion and/or from salmon embryo.These are more Peptide pollutant seems to have played inhibition effect in the catalysis of the interior Pepsin (zonase) of fish-egg, because their removal Add the catalytic activity of the interior Pepsin (zonase) of fish-egg.Also, their presence disturbs the interior albumen of the fish-egg The first step of the Edman- sequencings of ferment (zonase) product.In this 3rd purification step, the fish-egg of two kinds of forms it is interior The mode that Pepsin (zonase) is attached on column matrix seems have some different.However, two kinds of forms are in their N- There is similar amino acid sequence end.

The interior Pepsin (zonase) that fish-egg is determined from the partial amino-acid series of the CNBr- polypeptides produced is made For independent albumen.The enlightenment obtained from structural analysis：The interior Pepsin (zonase) of fish-egg may have obvious catalysis With Binding Capacity domain, these can explain their sensitiveness to calcium-chelating agent, when it acts on macromolecular (physiology) substrate (be attached to repressed, therefore catalysis suppressed indirectly), also explains their sensitivities for serine protease inhibitor Property, when it acts on small substrate (catalysis is inhibited directly).

Embodiment 5

The interior Pepsin (zonase) and leucoagglutinin (leukolectin) of fish-egg are all pure from salmon artemia hatching solution Change is obtained.In order to improve the protein concentration of artemia hatching solution, salmon roe is transferred to before hatching in minimal amount of water.It can pass through Improve (room) temperature or by deoxidation (Oppen-Berntsen et al.1990, Aquaculture, 86, pp.417- 430) hatching of high level of synchronization is induced, which creates the interior Pepsin (zonase) and GAP-associated protein GAP height of rough fish-egg The preparation of concentration.

The initial purification of the interior Pepsin (zonase) of fish-egg, which is related to filter by gauze (cheese cloth), hatches Salmon roe.The filtrate be defrosted and applied to further protein purification before, the several years can be frozen, and do not have fish The interior Pepsin (zonase) of ovum is significantly degraded.The fact that significantly simplify the production of parent material, the starting material Expect the interior Pepsin (zonase) and GAP-associated protein GAP of the fish-egg for purifying salmon, including leucoagglutinin (leukolectin)。

Next, optional step, which is related to, to be added to 4M urea by albumen filtrate and is adjusted, to separate salmon roe The fragment of shell, this allows to centrifuge (15,000g rotating speed twice, centrifuges 15min) by the broken of salmon chorion by low velocity Piece is removed together with the fragment of external source.The material does not show the phenomenon for blocking pillar, and this is through different from above-mentioned The characteristic for the crude product that method is prepared.The rough albumen preparation is applied to purify by traditional chromatographic technique.

Hatch liquid in leucoagglutinin (leukolectin) can together with fish-egg interior Pepsin (zonase) It is separated.From by interior Pepsin (zonase) preparation (as described above) of partially purified fish-egg, leucoagglutinin (leukolectin) it can be separated by exclusion chromatography (exclusion chromatography), because fish-egg Interior Pepsin (zonase) native form be much larger than leucoagglutinin (leukolectin).Separate, make for the first time With the pillars of Superdex 16/60, then, affinity chromatography benzamidine-agarose column (Benzamidine- can be passed through Sepharose columns) remove the interior Pepsin (zonase) of fish-egg.

Prepared for large batch of, be also applicable using ultrafiltration, because the day of the interior Pepsin (zonase) of fish-egg Right form is substantially not passed through the milipore filter that volume exclusion is 100kDa unlike leucoagglutinin (leukolectin).

The buffer solution used is millimole (millimolar) Tris, and its pH is about neutral or alkalescent (pH 7.5-8.5), it contains 5mM NaCl.Leucoagglutinin (leukolectin) is found the interior Pepsin with fish-egg (zonase) together co- purifying.The volume of the novel protein, is estimated under natural endowment by chromatography, only less than 30kDa.

The MW of the agglutinin is estimated in 25-30kDa or so.The pH estimations of the agglutinin of similar salmon are about 6.5.See The pIs (Riste is not published) observed, its ovum all agglutinin (salmon perivitelline lectins) in salmon PH value range is 6.5-6.9, its agglutinin (salmon leukocytic lectins) (agglutinin in salmon leucocyte Be identified by Western blotting technology) in pH value range in 6.4-6.6.

Embodiment 6

The research for evaluating the security and validity of the day cream for employing above-mentioned formula and the cosmetics of eye cream form is gone through When 12 week.40 female subjects are picked altogether, and the range of age is at 37-60 Sui.This 40 participants are fully completed The research.

Under conditions of the research, test material is identified in the way of day cream and eye cream, show its improve facial fine rule, Wrinkle, pigmentation, the colour of skin are uniform, skin transparency, cutis laxa degree and coarse sense of touch, by the use in 12 week Phase.In addition, the test material can be received well, when being applied on skin, it, which does not show, causes obvious corium to stimulate Possibility.

Daily using twice, the subsequent the 2nd, it is observed within 6 and 12 weeks, is determined using Packman and Gans score The lines on surface of face statistically significantly improves, using intuitive analog scale (Visual Analog Scale (VAS)) Score to determine facial pigmentation, the colour of skin is uniform, and skin transparency, cutis laxa degree and coarse sense of touch are statistically notable Improvement.

During any post processing, for significance,statistical, stimulation (erythema, oedema, drying, thorn are not observed Bitterly, burning and tight/dry sensation) increase.

Embodiment 7

Anti-microbial preservative resistance is evaluated to produce in the cosmetic for employing the day cream of formula disclosed above and eye cream form The research of the validity of microorganism pollution in product, is carried out in 2 research cycles of 4 weeks.It is micro- that research method employs CTFA Biological guide, the 20th part, M-3, the preservation method of testing and USP34 of aqueous personal care's product, the 61st part, neutralize/ Remove antibacterial activity.Test organisms includes staphylococcus aureus (Staphylococcus aureus), Escherichia coli (Escherichia coli), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Candida albicans (Candida ) and aspergillus niger (Aspergillus niger) albicans.For the 2nd class product, the preservative is considered as in given the test agent In be effective, if a) viable bacteria content showed the reduction for being not less than 3.0log series (99.9%) at the 7th day, relative to first Begin to count, and do not increase in whole section of test phase, and b) content of viable bacteria and mould was not less than 1.0log series at the 7th day Reduce (90%), do not increase relative to initial count, and in whole section of test phase.

Result of study shows, what the day cream and eye cream met the 2nd category product receives standard.

Embodiment 8

A research has been carried out to determine to lead using the day cream of formula disclosed above and the test material of eye cream form The ability of blackhead is caused, by evaluating whether the test material causes blackhead, by the patch bar in closure of surrounding Repeatedly applied under part, relative to negative control (uncoated area).In order to verify this research, the lanolin alcohol of acetylation is used Make positive control.The test is carried out during the research cycle of 4 weeks.15 subjects altogether, the age between 22 to 45 years old it Between, it is selected and participates in this research.15 subjects are completed this research.The biopsy specimen of hair follicle is collected from test position Evaluation shows, test material and control material are brought out 0.5 overall evaluation fraction (the small cutin of 1-24%).Test material and the moon Property control the average ratio of micro- blackhead of each hair follicle be 1%, positive control is 6%.Test material and feminine gender are right According to the ratio of micro- blackhead of hair follicle there is no significant difference, but positive control is shown significantly relative to negative control More height ratio.

The research shows do not have to report negative experience during studying.Test material, which is not shown, causes blackhead Ability.The site that the site handled with each test material and the patch of undressed negative control are applied, it is each The ratio of micro- blackhead of individual hair follicle does not have statistical discrepancy.

Embodiment 9

Carried out one research with determine using formula disclosed above day cream and eye cream form test material it is true Prickle swashs and sensitive possibility.Test is carried out within the research phase of 8 weeks.The research enables 225 subjects.11 tested Person does not complete research, and the reason for not completing is unrelated with test material.215 subjects complete the research altogether.

By the way that the patch with test material is applied into the test zone being dried through isopropyl alcohol and being allowed to To be tested.The test material is applied to the back of the body (between shoulder blade) and makes it keep direct skin contact small up to 24 When.On Monday, Wednesday and Friday, patch are applied to same site, are used for total 9 times during inducing.This arrangement of time It is modified to allow the visit missed or vacation.If subject can not report in specified testDate, the test material Expect to use at continuous two days during inducing and/or one cosmetic day of last increase during inducing.The corium in these sites is quick Sense degree is given a mark by supervision technical staff, 24 hours after Tuesday and Thursday subject eliminate patch, and on Sunday Remove after patch 48 hours, unless the arrangement of time of patch described above is changed.

These sites are given a mark according to following scoring systems：

Corium score scale

0 without obvious dermoreaction

+ erythema is discovered reluctantly

Erythema gentle 1+

2+ clearly erythema

Erythema and oedema serious 3+

Erythema and oedema of the 4+ with foaming

If " 2+ " reacts or stronger there occurs, then the original position for being applied to close on by the test material Put.If there occurs " 2+ " reaction or stronger in new position, then subject will not be applied during the induction by patch again, But can be challenged in the proper time of the research.The score that patch applies position may have changed less than " 2+ ", no matter research Supervisor is handled.

Afterwards during the test of about 2 weeks, the patch of challenge is applied to the untreated test site before of back.24 is small Shi Hou, patch is removed by CRL technical staff and the corium in evaluation test site reacts.24 hours and 48 hours, test site quilt Reappraise.During the challenge of the research, it is shown that the subject of reaction may be called back, observe 96 hours.

Test crowd based on 214 subjects and under conditions of the research, eye cream test material does not show initiation Corium is stimulated or sensitive ability.

Embodiment 10

It is extra anti-to determine to the addition of using formula disclosed above and in the formula to have carried out a series of research Shining the corium of the day cream of index (SPF) composition and the test material of eye cream form stimulates and sensitive ability.SPF is defined as Amount of energy on the skin protected required for the minimum erythema of generation on untreated skin with producing minimum erythema institute The ratio of the amount of energy needed, calculating formula is as follows：

I. a research has been carried out to evaluate the efficiency for the sunscreen product for being combined with the day cream prepared using the above method, has led to Cross and SPF (SPF) is determined under the conditions of statistics using international SPF (SPF) method of testing (in May, 2006). The sunscreen formulas of one standard is detected simultaneously with test material.Standard is padimate oxygen/oxybenzone SPF Standard (Padimate O/Oxybenzone SPF Standard (COLIPA P2)) (COLIPA P2).Test/control material The site of application is, at back, and to be randomly provided with so that receiving the treatment dorsal area of single test material for institute It is different to have for subject.10 masculinity and femininity subjects altogether, the age is between 25 years old to 57 years old and basic upper body Body health, is selected and participates in this research.During whole research, the report without adverse events.

II. a research has been carried out to evaluate the sunscreen product for the SPF30 for being combined with the day cream prepared using the above method Efficiency, according to the UVA of Japanese cosmetic employer's organization (JCIA) protect examination criteria (01/01196), in statistics condition Under, determine ultraviolet light,long wave (UVA) sun protection factor (PFA).10 adults take part in this research altogether.One hernia arc Multiport sunlight simulator (Xenon Arc Multi-Port Solar Simulator) (150w, Model 15S, Solar Light Company, Philadelphia, PA), the ultraviolet with UVA and UVB wave bands (290-400nm) is continuously radiated Wave spectrum, it is used for the source of ultraviolet radiation and is filtered to provide a basic wave spectrum as sunlight.Using Following filter is to ensure that suitable wave spectrum is exported：Schott WG33513mm and UG1111mm.Light output (lamp Output) in each subject by UV intensity instrument (UV Intensity Meter) (Model PMA2100, Solar Light Company, Philadelphia, PA) test before measured in time.The wave spectrum distribution of the light output of sunlight simulator Annual correction is once.

Melanism is exposed to the taupe (brown-gray) observed after UVA on skin to the reaction of brownish black, is Because light-oxidation reaction, colourless melanin precursor is oxidized to form the melanin of coloring in the reaction.It is minimum lasting Melanism (MPPD) is defined as inducing UVA light radiation dosage necessary to such reaction on untreated skin.In test Before stage, the MPPD of each subject is the progressive grade exposed to the open air by UV light than several determinations.The increasing of the dosage of UVA radiation It is 2 to 4 hours after 1.25. radiation to double number, and the melanism situation in these sites is evaluated according to following scoring system.

0 is negative, does not react significantly；

± (minimum) melanism is discovered reluctantly；

1+ clearly (medium) melanism；

2+ is significant or melanism of boundary clear.

It is drawn to scale with operation marking pen between subject's shoulder blade and waistline, on the back of center line both sides as 5*10cm Test site profile.These regions are designated as test material or sun-proof standard.Neighbouring site is designated as not locating MPPD measure is carried out on the skin of reason simultaneously.After products application, each test zone is subdivided into about 1 square centimeter of position Point, for determining that continuous UVA light exposes to the open air.

One 0.1ml parts of test material or standard are applied to suitable test site and make it fifty-fifty using fingerstall Whole site is extended to (equivalent to 2 μ l/cm²).Start loci after use, between 15 minutes to 30 minutes to be radiated.Base It is each processing in the MPPD and test material or the expection PFA of standard of the measure of the not protected skin determined before Site selects exposure time.All test sites were evaluated in 2 to 4 hours to determine the lasting melanism of minimum after exposure Reaction.

UVA protective factors (PFA) are defined as producing the time quantum required for a MPPD on the skin protected Divided by the ratio of the time quantum required for a MPPD is produced on untreated skin, calculating formula is as follows：

Standard error is needed within the 10% of test value.

UVA protection level is determined by the integer value of the PFA values in the site of radiation treatment.Because all PFA values are all big In or equal to 2, protection level is determined using following sorting technique：

The average UVA protective values that the test material SPF30 submitted day cream is shown in 10 subjects are PFA6.5.

III. carried out one research with evaluate using above-mentioned formula be formulated containing the sun-proof of wide spectrum sun-screening function Active matter day cream test material, by according to FDA (Friday of federal register/Vol 76, No 117/, June 17,2011/ Rule and regulation) method that provides determines its crucial wavelength.

The absorbance of sunscreen product is the summation that whole UV wavelength is crossed over since 290nm, until total sum reaches 90% The sunscreen product UV wavelength range (290-400nm) total absorbance.The summation of absorbance reaches total absorbance Wavelength when 90% is defined as critical wavelength (critical wavelength) and is considered as the wide cut of preventing coating protection Weights and measures.When critical wavelength is more than 370nm, the sunscreen product of a pith of the absorbance with them in UVA It is classified as wide spectrum (broad spectrum) sunscreen product.

Determine spectral-transmission favtor (Spectral of each wavelength in whole UV spectral regions (290-400 nanometers) transmittance ).The value of a transmissivity is measured at interval of 1nm.

The crucial wavelength of day cream of the test material with SPF is 371.00nm, and the wavelength meets " wide spectrum " label institute It is required that minimum 370nm critical wavelength standard.

Carry out a similar testing research and contain wide spectrum sun-screening function using what above-mentioned formula was formulated to evaluate Sunscreen actives thing day cream test material, by determining its external UVA protection factor according to the COLIPA guide of 2011 (UVA-PF), SPF/UVA-PF ratios and the critical wavelength of the day cream.

Any invention document or publication referred in this specification is all incorporated to by reference herein, the journey being incorporated to Degree specifically and is individually illustrated to be merged in by reference such as any one single publication.

Above-mentioned specification is particular implementation of the present invention for example, it should not turn into the limit for implementing the present invention System.

Claims

1. a kind of cosmetic composition, it is included：

It is that hatch fish roe is obtained and including activated protein be interior Pepsin Protein Separation thing；And

Natural extracts containing unrighted acid and sterols material；

Wherein described activated protein constitutes the 3.4 × 10 of described composition total weight^-7% to 3.4 × 10^-4%.

2. cosmetic composition according to claim 1, it is characterised in that described composition includes emulsifying agent, solubilising Agent or combinations thereof.

3. cosmetic composition according to claim 2, it is characterised in that described composition includes emulsifying agent and is water Bag oil form emulsion.

4. cosmetic composition according to claim 2, it is characterised in that described composition includes emulsifying agent and is oil Bag water form emulsion.

5. cosmetic composition according to claim 1, it is characterised in that described natural extracts carry for algae Take thing.

6. cosmetic composition according to claim 1, it is characterised in that described natural extracts carry for brown alga Take thing.

7. cosmetic composition according to claim 1, it is characterised in that described natural extracts are from plant Leaf, stem, bud, root or their compositions of thing.

8. according to cosmetic composition according to any one of claims 1 to 7, it is characterised in that also including ascorbic acid Portugal Glucosides.

9. according to cosmetic composition according to any one of claims 1 to 8, it is characterised in that described fish-egg is salmon Ovum.

10. according to cosmetic composition according to any one of claims 1 to 9, it is characterised in that described cosmetic combinations When thing is applied to skin, improves skin firmness, smoothness or both and be improved.

11. a kind of be used to improve the cosmetic composition of skin appearance, described composition is emulsion, including：

The Protein Separation thing that hatch fish roe is obtained, described Protein Separation thing is used as activated protein including interior Pepsin；

Natural extracts containing unrighted acid and sterols material；

Emulsifying agent；And

Antimicrobial,

12. cosmetic composition according to claim 11, it is characterised in that described fish-egg is salmon roe.

13. cosmetic composition according to claim 11, it is characterised in that described cosmetic composition includes anti-bad Hematic acid glucoside.

14. cosmetic composition according to claim 11, it is characterised in that described natural extracts are derived from Red algae, brown alga or tufted cyanobacteria.

15. the cosmetic composition according to any one of claim 11~14, it is characterised in that described cosmetics group When compound is applied to skin, improves skin firmness, smoothness or both and be improved.

16. a kind of method by improving the improvement skin appearance of skin firmness, smoothness or both, described method includes Cosmetic composition described in usage right requirement any one of 1~15 is on human skin, at least three times a week, wherein institute The method stated includes raising skin firmness, smoothness or both and is improved.